Hexamethylene bisacetamide (HMBA) induces cellular differentiation (e.g. Wang et al., Chin Med Sci J 2002, 17, 27–31), and has been shown to modulate the expression of a wide variety of genes (e.g. Zhang et al., August 2004, Int J Biochem Cell Biol, 36, 1613–23). It has also been reported to activate viruses, such as HSV (Yura et al., J Natl Cancer Inst 1991, 83, 186–9; McFarelane et al., J Gen Virol 1992, 73, 285–92) and HIV (e.g. Vlach et al., J Gen Virol. 1993, 74, 2401–8), and to inhibit human vascular smooth muscle cell (VSMC) proliferation (e.g. Ishikawa et al., Coron Artery Dis. 1997, 8, 28–32). Accordingly, HMBA and related compounds have been intensively studied as anti-cancer agents (e.g. Marks et al. C R Acad Sci III. 1999, February-March;322(2–3):161–5).
One HMBA-inducible protein of VSMCs, HEXIM1 (Kusuhara, et al. 1999, Biomed. Res. 20, 273–279), also known as HIS1, CLP1, MAQ1 and EDG1, has been reported to inhibit NFkB-activated transcription in VSMCs, and overexpressed HEXIM1 has been proposed as a strategy to therapeutically inhibit VSMC proliferation (Ouchida et al., Genes Cells (2003) 8:95–107). Independently, CLP-1 (cardiac lineage protein-1), the chicken and mouse ortholog of HEXIM1, has been suggested to play a role in cardiogenesis (Ghatpande et al., 1999, Dev Biol 208, 210–221; Ghatpande et al., 1999, Mol Cell Biochem. 96, 93–97; Huang et al., 2002, Gene 292, 245–259). Overexpression of CLP-1 inhibits the cardiac MLC-2v gene transcription (Huang et al., Mech Dev., 2004, 121:559–572; 2002). Also independently, increased expression of HEXIM1 has been reported to inhibit transcriptional activity in breast endothelial cells (Wittmann et al., Cancer Res. (2003) 63:5151–5158; Montano et al., U.S. Pat. No. 6,753,418; wherein HEXIM1 is referred to as EDG1). Also independently, Michels et al. (EMBO J., 2004, 23:2608–2619; and Mol Cell Biol., 2003, 23:4859–4869; wherein HEXIM1 is referred to as MAQ1) report that in HeLa cells, a HEXIM1-7SK RNA complex can bind CDK9/CyclinT (P-TEFb) as part of the 7SK snRNP.
P-TEFb serves not only as a general transcription factor but also a specific cellular cofactor for the HIV-1 Tat protein. Preventing Pol II from stalling is essential for HIV-1 transcription, during which P-TEFb is recruited to the nascent mRNA by Tat through formation of a ternary complex containing P-TEFb, Tat, and the HIV-1 TAR RNA, a stem-loop structure formed by the 5′ end of the nascent viral transcript. Once recruited, P-TEFb phosphorylates the CTD and stimulates transcriptional elongation to produce the full-length HIV-1 transcripts.
Besides HIV-1 replication, the activation of P-TEFb also promotes cardiac hypertrophy, a disease characterized by the enlargement of cardiac myocytes. P-TEFb activity has been shown to be limiting for normal cardiac growth. Hypertrophic signals induce the release of 7SK and an increase in the cellular level of active, 7SK-free P-TEFb (Sano et al., Nat. Med., 2002, 8:1310–1317; Sano et al., Cell Cycle. 2003, 2, 99–104).
Practical utilization of HEXIM1 has been limited without any functional assignment for the protein. We disclose that P-TEFb is inhibited by HEXIM1 in a process that specifically requires 7SK for mediating the HEXIM1:P-TEFb interaction. We demonstrate that cellular transcription can be directly manipulated by increasing or decreasing HEXIM1 activity in the cell. More particularly, we show that HEXIM1 inhibits CDK9/Cyclin T, and can be used to inhibit cardiac hypertrophy and inhibit HIV replication. The disclosed methods for targeting fully differentiated cells, particularly non-proliferating cell types, such as hypertrophic cardiac myocytes provides unexpected therapeutic venues.
Relevant Literature
We have reported aspects of the present invention in Chen et al., J Biol Chem. 2004 Feb. 6;279(6):4153–60. Epub 2003 Nov. 19; Yik et al., Mol Cell Biol. (2004) 24:5094–105; and Yik et al., Mol Cell (2003) 12:971–982.